Cytokines control many important cellular responses by activating intracellular signaling cascades including the Jak-Stat pathway that leads to the transcriptional activation of specific genes. The regulation of cytokine signaling requires a delicate balance between biochemical events that activate and amplify the signals initiated by the cytokine receptor-associated Jaks, and the mechanisms that inactivate the Jaks and thereby attenuate the signals. Although biochemical mechanisms responsible for initiation and amplification of cytokine signals have precisely been defined in the recent years, negative regulation of cytokine signaling remains poorly understood. The long term goal of this application is to investigate cellular mechanisms responsible for the negative control of cytokine signaling, using the IL-4 signaling system as a paradigm. IL-4 plays multiple roles in host defense and immunity. It regulates growth and differentiation of B and T cells and is recognized as an important determinant of the nature of the immune response through the promotion of humoral response at the expense of cell mediated immunity. IL-4 activates two intracellular signaling cascades in target cells: the IRS-PI3-Kinase pathway that leads to cell proliferation, and the Jak-Stat pathway that induces the transcription of a number of specific genes. Both pathways are activated by Jak-mediated tyrosine phosphorylation of specific proteins. Our preliminary work has identified a protein tyrosine phosphatase Shp-1 and two suppressor of cytokine signaling (SOCS)-family proteins SOCS-1 and SOCS-3 as negative regulators of IL-4-dependent signal transduction. In this application we propose the hypotheses that (i) IL-4 receptor activation is negatively controlled by Shp-1 and additional protein tyrosine phosphatases(s) (PTPs) at an early phase, and (ii) IL-4-inducible proteins SOCS-1 and SOCS-3 down-regulate the receptor activation in late phase, by acting through a feedback loop. We will test these hypotheses by pursuing the following specific aims. (1) To define the roles of PTPs in the negative regulation of IL-4 signaling we will identify Shp-1 substrate(s), investigate the structural basis of the functional difference between Shp-1 and Shp-2, and identify additional PTPs that associate with the IL-4Ra complex and inhibits receptor activation. (2) To investigate the roles of specific SOCS-family proteins in the negative regulation of IL-4 signaling we will define the structural basis and molecular mechanisms of SOCS-1- and SOCS-3-mediated inhibition, and determine how the constitutive or induced expression of SOCS-1 and SOCS-3 prior to the initiation of IL-4 receptor activation affects the signaling process.